Transcript Cell, Vol. 122, 579–591, August 26, 2005, Copyright ©2005

A Role for Proapoptotic BID
in the DNA-Damage Response
Zinkel S.S. et al., (2005) Cell 122,579-591
銘傳大學
生物科技系
學生：徐鉦竤
指導教授：陳秀儀老師
Introduction
BH
Introduction
Introduction
1.The BH3-only member BID interconnect the intrinsic pathway
and extrinsic Death receptor pathway .
2.BID in maintaining myeloid homeostasis and suppressing
leukemogenesis.
3.In a resting cell, BID is predominantly cytoplasmic. Following
TNF or Fas treatment,BID is cleaved by caspase 8 in an
unstructured loop, facilitating its translocation to the mitochondr
1.BID有穩定染色體的功能
material
Cell line:
Hox11-immortalized premalignant myeloid progenitor cell line(MPCs)
HOX 11 gene會造成t (7:10) (q35;q24) or t(10:14)(q24;qll)的
translocations.與T-cell leukemia 和 lymphoid neoplasias有關。
欲證明BID的存在時會抑制此現象發生。
Method
Mataphase Spreads
wild-type and Bid−/− MPCs
mytomycin C, 24hr
arrested in metaphase 0.1 mg/ml of colchicine,
fixed
visualized
Giemsa staining
Result
Fig.1
Result
藉由每個細胞中所增加的breaks數來計數in 50 metaphase spreads
Fig.1
Each chromosomes break was given a score of +1
Each tri- or quadriradial was given a score of +2
total 50 metaphase spreads
Result
Fig.1
Increased chromosal instability is a general feature of Bid dificiency
Result
Fig.1
Each chromosomes break was given a score of +1
Each tri- or quadriradial was given a score of +2
total 50 metaphase spreads
Result
Bid-/- MPCs display increased sensitivity to DNA-damage agents
Fig.2
Result
Bid−/− primary activated T cells in response to IR (data not
shown), a cell type that is less dependent upon the BIDmediated
mitochondrial
amplification
loop for death (Scaffidi et al., 1998).
Fig.2
Result
Fig.3
Aphidicolin is Reversible inhibitor of eukaryotic nuclear DNA replication.
Result
To verify that this S phase phenotype is attributable to the absence of BID.
Fig.3C
Fig.3D
Conclusion
The death function of proapoptotic BCL-2 family member
is localized to the BH3 domain. To determine whether an
intact BH3 domain is required for the BID deficient S phas
defect.
Result
Fig.4
Result
Fig.4
Result
Fig.4
A region of BID distinct from its proapoptotic BH3 domain mediates
the BID’s role downstream of DNA damage induced by replicative
stress.
To further elucidate the kinase responsible for
Regulation of BID phosphorylation, the authors
evaluated a series of MEFs deficient for the DNA
repair kinases ATM, ATR,and DNA-PKcs.
MEF display significant genotypic and phenotypic variations depending
on the strain and the genetic background of the mice they were isolated
from as well as the immortalization strategy used.
Fig.6A
BID has at least two ATM/ATR/DNA-PKcs consensus
Phosphorylation sites at residues S61 and S78
Result
Fig.S6
Wild-type MPCs
Result
Fig.6B
1mM 10mM
1 μM 10 μM
1 μM 10 μM
1mM 10mM
1 μM 10 μM
1 μM10 μM
ATM/ATR/DNA-PKcs是在同一的位置對BID的磷酸化，
表示BID磷酸化是受這些DNA repair kinase所調控。
Result
Fig.6E
MEFs
缺少ATM，BID磷酸化幾乎完全消失
Result
Fig.6H
Fig.S8
甚至部份的ATR或DNA-PKcs失活，BID還是可以被磷酸化
The S Phase Role of BID Is Mediated by Phosphorylation
at Position 78
Result
Fig.7B
在Bid-/- MPCs中的Bid S78A mutant無法恢復由Aphidicolin
造成的intra-S phase arrest，表示BID的S78A可能在intra-S
Phase checkpoint起作用。
Conclusion
Conclusion
1. BID plays an unexpected role in the intra-S phase
checkpoint downstream of DNA damage.
2.BID有穩定染色體的功能
3.A Functional BID BH3-Domain Is Not Required
to Rescue S Phase Accumulation.
4.this role is mediated through BID phosphorylation by
the DNA-damage kinase ATM.
5.ATM/ATR在BID的S78A磷酸化可以調控intra-S phase
checkpoint
Thank you for your attention
Giemsa staining
A mixture of glycerin, methanol, methylene azure, and eosin used to
stain chromosomes.
Subcellular Fractionation
Cells
sucrose lysis buffer
lysed
centrifuged at 1200 RPM for 5 minutes
Supernatant
centrifuged at 8000 RPM
12minutes
cytosolic fraction
pellet
low salt buffer
resuspend
an equal volume of high salt buffer
centrifugation,
at 12,000 RPM for 10 minutes
The soluble nuclear fraction
pellet
10 mM HEPES pH 8.0
1.5 mM MgCl2
10% glycerol
resuspend
with Benzonase nuclease (Novagen)
digested
centrifugation at 12000 RPM
The nuclear pellet fraction
Result
Fig.5C
Subcellular fractionation of MPCs at 2 hr following hydroxyurea
treatment (1 mM). BID is found in the insoluble chromatin fraction
following hydroxyurea.
Manganese superoxide dismutase (MnSOD)
O2- 
H2O2 +
O2 (mitochondria)
Under normal physiological conditions, mitochondria are the major
source of O2-  production. Numerous studies have indicated that
MnSOD plays an important role in preventing cells from oxidative
stress and inhibiting tumorigenicity.
The human RAD9 gene partially complemented the hydroxyurea
sensitivity, radiosensitivity, and checkpoint defects of rad9-null
mutant cells. In vivo, the human RAD9 protein was phosphorylated in
response to DNA damage, suggesting that it participates in a DNA
damage-inducible signaling pathway.
Method
Cell cycle analysis
(propidium iodide staining)
One million cells
Resuspended in 200μl Krishan’s reagent
Incubated
15min,
Room Temperature,
in the dark
Analyzed on a Becton-Dicknson FACS machine
using Flojo analysis software
background
Krishan’s reagent
•
•
•
•
0.1% NaCitrate
0.03%NP40
0.05mg/ml propidium iodide
0.02mg/ml RNase a
Method
BrDU analysis
Cells were incubated with 10 μl BrDU for 45min
Washed
Incubated with 100mM hydroxyurea or
0.1 μM aphidicolin
Murine Stem Cell Virus retroviral vector (MSCV)
1.The Murine Stem Cell Virus (MSCV) vectors were derived from
the Murine Embryonic Stem Cell Virus (MESV).
2. The vectors achieve stable, high-level gene expression in
hematopoietic and embryonic stem cells through a specifically
designed 5' long terminal repeat (LTR).
293T cells
293T is a human embryonic kidney cell line commonly used
for transfection assays.The expression of the large T antigen
in the cell, plasmids with SV40 origin of replication can be
transiently transfected and give extremely high
levels of expression of AP fusion proteins.
In Vitro Kinase Assays
293T cells
24 hours post transfection
10Gy IR for 30 minutes
treated with 10Gy IR , 30 minutes
harvested and lysed in TNE buffer
adding 30 μl of kinase buffer
Kinase assays
incubating for 30 minutes at 30°C
293T cells
293T is a human embryonic kidney cell line commonly used
for transfection assays.The expression of the large T antigen
in the cell, plasmids with SV40 origin of replication can be
transiently transfected and give extremely high
levels of expression of AP fusion proteins.
Fig.6I
Bid is a substrate for ATM and ATR in vitro
flox
An ATR targeting construct containing a duplicated exon 2 as well as
an exon 2 disrupted in frame with the coding region of the neomycin
resistance gene and polyadenylation sequence was created using ATR
genomic DNA cloned from a lambda genomic library.
Cre system
the system has been developed as a means to efficiently edit and
manipulate the genome. It has been successfully applied in studies
of yeast, plants, mammalian cell culture and mice.1 Targeted
mutations, transgenics, allelic modification, deletions, and
chromosomal aberrations can all be produced using variations of
a common reaction.